Outbreaks of influenza A virus continue to cause widespread morbidity and mortality worldwide. In the United States alone, an estimated 5 to 20% of the population is infected by influenza A virus annually, causing approximately 200,000 hospitalizations and 36,000 deaths. The establishment of comprehensive vaccination policies has been an effective measure to limit influenza morbidity. However, the frequent genetic drifting of the virus requires yearly reformulation of the vaccine, potentially leading to a mismatch between the viral strain present in the vaccine and that circulating.
Influenza A virus consists of 9 structural proteins and codes additionally for two nonstructural NS1 proteins with regulatory functions. The segmented nature of the viral genome allows the mechanism of genetic reassortment (exchange of genome segments) to take place during mixed infection of a cell with different viral strains. The influenza A virus is classified into various subtypes depending on the different hemagglutinin (HA) and neuraminidase (NA) viral proteins displayed on their surface. Influenza A virus subtypes are identified by two viral surface glycoproteins, hemagglutinin (HA or H) and neuraminidase (NA or N). Each influenza virus subtype is identified by its combination of H and N proteins. There are 16 known HA subtypes and 9 known NA subtypes.
Influenza virus is a negative-sense segmented RNA virus that can infect many animal species including human. The replication of influenza genome by the viral coded RNA dependent RNA polymerase is an error prone process generating progenies with varied genetic sequences at all times. Viable viruses with genetic alterations are designated “antigenic drift” mutants. The segmented nature of the viral genome and the possibility to infect different animal species could produce “antigenic shift” mutants (P. K. Cheng et al., Emerg. Infect. Dis. 15, 966 (2009)). Under desirable conditions, dominant variants may become prominent pathogens for human or animals. The multi-step selection processes leading to mutant evolution are not completely understood (L. Cohen-Daniel et al., J. Clin. Virol. 44, 138 (2009); R. Wagner, M. Matrosovich, H. D. Klenk, Rev. Med. Virol. 12, 159 (2002)). Whereas vaccines are often used for the prevention of influenza virus infections, the most useful therapies for the treatment of influenza infections involve administration of Tamiflu® (the phosphate salt of oseltamivir ethyl ester, Roche Laboratories, Inc.) and Relenza® (zanamivir, Glaxo Wellcome, Inc.). (N. J. Cox, J. M. Hughes, N. Engl. J. Med. 341, 1387 (1999)). Oseltamivir and zanamivir are viral sialidase (neuraminidase) inhibitors that prevent the release and dispersal of progeny virions within the mucosal secretions and thereby reduce viral infectivity. Neuraminidase (NA), a glycoprotein expressed on the influenza virus surface, is essential for virus replication and infectivity by breaking the linkage between the progeny virus from the surface sialo-receptor of host cells. Thus, inhibition of NA by the structure-based strategy has been applied in discovery of anti-influenza drugs.
Zanamivir (Relenza™) (von Itzstein, M. et al. Nature 1993, 363, 418. Dunn, C. J.; Goa, K. L. Drugs 1999, 58, 761.) is a popular drug for the treatment of influenza. Tamiflu is a prodrug that is readily hydrolyzed by hepatic esterases to give the corresponding oseltamivir carboxylic acid as the active inhibitor to interact with three arginine residues (Arg118, Arg292 and Arg371) in the active site of viral neuraminidase (NA). (von Itzstein, M. et al. Nature 1993, 363, 418. Lew, W. et al. Curr. Med. Chem. 2000, 7, 663. Russell, R. J. et al. Nature 2006, 443, 45.) Both oseltamivir and zanamivir inhibit influenza virus NA that is essential for virus propagation by cleaving the linkage between the progeny virus from the surface sialo-receptor of host cells. The NA inhibitors are designed to have (oxa)cyclohexene scaffolds to mimic the oxonium transition-state in the enzymatic cleavage of sialic acid (N-acetylneuraminic acid), the outmost saccharide on the cell surface glycoprotein for binding with the active site of viral NA. To accommodate the binding with oseltamivir carboxylic acid, an induced fit of the NA to create a large hydrophobic pocket is needed for the 3-pentyl side chain. (Collins, P. J., et al. Nature 2008, 453, 1258.) In comparison, zanamivir is less susceptible to the newly evolved resistant viruses than oseltamivir phosphate. In the absence of the need for generating the hydrophobic binding pocket, the inhibition potency of zanamivir to the NA mutant (e.g. the clinically relevant H274Y mutant) is unchanged.
Influenza A (H1N1) viruses bear a oseltamivir resistance conferring amino acid change of histidine to tyrosine at position 274 (H274Y) of the neuraminidase (NA) protein. The 2008 surge of the oseltamivir resistant H274Y mutants in seasonal H1N1 (A. Moscona, N. Engl. J. Med. 360, 953 (2009)) was puzzling because the increases of these mutations are not correlated to oseltamivir usage in many of the H274Y prevalent areas (J. Mossong et al., Antiviral Res. 84, 91 (2009); M. Jonges et al., Antiviral Res. 83, 290 (2009)). In addition, the H274Y oseltamivir resistant pandemic H1N1 (A. Gulland, Br. Med. J. 339, b4975 (2009)) and the H5N1 mutants (Q. M. Le et al., Nature 437, 1108 (2005)) are reported in patients suggesting that these mutants could impact influenza therapy options (I. Stephenson et al., Clin. Infect. Dis. 48, 389 (2009)).
Many derivatives of zanamivir have been prepared by modification at the glyceryl moiety scaffolds to mimic the oxonium transition-state in the enzymatic cleavage of sialic acid. The phosphonate group is generally used as a bioisostere of carboxylate in drug design. (White, C. L. et al. J. Mol. Biol. 1995, 245, 623. Schug, K. A.; Lindner, W. Chem. Rev. 2005, 105, 67. Streicher, H.; Busseb, H. Bioorg. Med. Chem. 2006, 14, 1047.) In comparison with the carboxylate-guanidinium ion-pair, a phosphonate ion will exhibit stronger electrostatic interactions with the guanidinium ion. Thus, the zanamivir phosphonate congener is expected to have more potent against the neuraminidases of H1N1 and H5N1 viruses, even the H274Y mutant. The enhanced affinity may be attributable to the strong electrostatic interactions of the phosphonate group with the three arginine residues (Arg118, Arg292 and Arg371) in physiological conditions.
Solution-phase neuraminidase inhibition assays normally use the fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-D-acetyl-neuraminic acid, which is cleaved by neuraminidase to yield a fluorescent product that can be quantified using a fluorometer (Potier et al., Anal. Biochem. 94:287-296 (1979)), however, this assay method is not amenable to a high-throughput format. In addition, due to the fast emergence of resistant viral strains (see McKimm-Breschkin, J. L. Antiviral Res. 2000, 47, 1-17), there remains a need to find new influenza neuraminidase inhibitors.